A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene
Graphical abstract
Introduction
Not only aging, but also central nervous system (CNS) diseases, such as Alzheimer disease (AD), one of the most devastating neurodegenerative diseases, progressively reduce brain functions. Impairment of cognitive functions characterized by learning and memory loss is a specific feature in aging and AD patients (Terry and Davies, 1980). Degeneration of presynaptic cholinergic system is a major cause of cognitive deficit in AD patients (Coyle et al., 1983, Whitehouse et al., 1982) and is known to be attributable to the decreased activity of choline acetyltransferase (ChAT), which is necessary for acetylcholine (ACh) synthesis (Kasa et al., 1997, Terry and Buccafusco, 2003).
AF64A is a choline analog that is taken up only by the high-affinity choline transport system into cholinergic neurons (Fisher et al., 1982) and causes alterations in mRNA expression and activity of ChAT (Bessho et al., 2008, Fan and Hanin, 1999, Yamazaki et al., 1992). Therefore, AF64A administration was found to decrease the release of ACh leading to cognitive impairments, including memory and learning deficits, a salient characteristic of AD (Abe et al., 1993, Bessho et al., 2008, Yamazaki et al., 1992).
Thus far, AD therapy has relied on compounds that increase the ACh concentration, such as acetylcholinesterase (AChE) inhibitors, as well as N-methyl-d-aspartate (NMDA) receptor antagonists (Bessho et al., 2008, Eleti, 2016, Musial et al., 2007, Takashina et al., 2008, Terry and Buccafusco, 2003). In addition, enhancers of mRNA expression of ACh-synthesizing enzyme ChAT were used to recover impaired learning and memory functions (Egashira et al., 2003, Karakida et al., 2007, Shin et al., 2016, Wang et al., 2000). In particular, ginsenoside Rb1 was used to increase the expression of ChAT mRNA (Qi et al., 2011), thereby increasing synaptosomal choline uptake and the ACh release (Benishin, 1992, Benishin et al., 1991, Scholey et al., 2010). In addition, in our previous studies, we demonstrated that transplantation of neural stem cells (NSCs) encoding ChAT gene (F3.ChAT) markedly recovered cognitive function of animal models of AD and aging (Park et al., 2012a, Park et al., 2013).
Silk proteins from silkworm (Bombyx mori) are well known to have pharmacological activities such as anti-diabetic effect (Lee et al., 2007, Park et al., 2002). When degraded by enzymes, silk proteins yield specific sizes or compositions of silk peptides (SPs) that show diverse bioactivities, including anti-diabetic, hypocholesterolemic, and antioxidative actions (Kato et al., 1998, Kim et al., 2008, Lee et al., 2007, Zhaorigetu et al., 2003). In addition, silk amino acids (SAAs; composed of 19 amino acids) obtained from full degradation of silk proteins via acidic (HCl) hydrolysis have been reported to prevent oxidative injures of tissues, thereby enhancing physical stamina (Shin et al., 2009a, Shin et al., 2009b). Another study reported that SAA preserved dopaminergic nerves in the Parkinson disease (PD) model animals to improve the animals' movement disorders (Kim et al., 2011). Still another study reported that SP improved cognitive function of rats with aging brain facilitated by D-galactose (Park et al., 2011).
Such results led us to investigate the cognition-enhancing effect of an enzyme-degraded silk peptide prepared via the incubation process of silk proteins with Protease N and Neutrase (SP-NN) in AD model animals. In order to elucidate the underlying mechanisms, the effect of SP-NN on the expression of ChAT mRNA was assessed in F3.ChAT neural stem cells and the active amino acid sequence was identified using HPLC-MS.
Section snippets
SP preparation
Freeze-dried silk peptide-NN (SP-NN; Worldway code: PF-B6) was obtained from Worldway Co., Ltd. (Sejong, Korea). To prepare SP-NN, silk proteins were incubated for 12 h with Protease N (150,000 U/g; Amino G from Bacillus subtilis) plus Neutrase (160,000 U/g; Bacillus amyloliquefaciens). In short, the cocoon were dissolved at 115 °C for 60 min in the KCl solution and then sterilized at 90 °C for 30 min. After being cooled to 50 °C, the proteins were degraded using the above enzymes at pH 5.8 and
Isolation and purification of the active compounds
SP-NN and each fraction separated by gel filtration chromatography were assessed for their effects on the ChAT mRNA expression in F3.ChAT cells using a RT-PCR. Twenty four-hour incubation of the cells with SP-NN enhanced the ChAT expression 1.42 and 1.78 times the control level at 10 and 100 μg/ml, respectively (data not shown). Since the 3rd fraction [SP(3)] of gel filtration chromatography of SP-NN was the most potent, the fraction was subjected to semi-preparative HPLC for isolation, and nine
Discussion
Cognitive functions are known to comprise the acquisition (learning) of information and the retention of acquired memory. It is well known that ACh secreted from the central cholinergic nervous system is involved in the formation of short-term memory (acquisition). Long-term memory is formed through the consolidation of acquired information through long-term potentiation of glutamatergic nervous system after activation of the NMDA receptors (Musial et al., 2007). Therefore, deterioration of the
Conflict of interest
The authors declare that they have no actual or potential competing financial interests.
Transparency document
Acknowledgments
This research was supported by High Value-Added Food Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA; grant number 113034-3) and by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2015R 1A 6A 1A 04020885).
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